We thank the laboratorians, epidemiologists, and affiliates with public health departments for sample submissions to the Centers for Disease Control and Prevention. The findings and conclusions in this report are those of the authors and do not necessarily represent the official position of the Centers for Disease Control and Prevention. Use of trade names and commercial sources are for identification only and do not imply endorsement by the Centers for Disease Control and Prevention.

While the IHC protocol was performed on formalin-fixed tissues, which inactivates RABV if present, appropriate biosafety protocols should be properly followed. All biosafety procedures are described in the Biosafety in Microbiological and Biomedical Laboratories (BMBL) 5th Edition (https://www.cdc.gov/biosafety/publications/bmbl5/index.htm), including wearing proper personal protective equipment (PPE), and vaccination requirement as described12. In addition, proper containment and handling of haza...

<ol>
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E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescent+antibody+staining+of+street+and+fixed+rabies+virus+antigens.">Fluorescent antibody staining of street and fixed rabies virus antigens.</a> Proceedings of the Society for Experimental Biology and Medicine. 98 (2), 219-223 (1958).</li><li>Lembo, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+a+direct,+rapid+immunohistochemical+test+for+rabies+diagnosis.">Evaluation of a direct, rapid immunohistochemical test for rabies diagnosis.</a> Emerging Infectious Diseases. 12 (2), 310-313 (2006).</li><li>Fekadu, M., Greer, P. W., Chandler, F. W., Sanderlin, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+the+avidin-biotin+peroxidase+system+to+detect+rabies+antigen+in+formalin-fixed+paraffin-embedded+tissues.">Use of the avidin-biotin peroxidase system to detect rabies antigen in formalin-fixed paraffin-embedded tissues.</a> Journal of Virological Methods. 19 (2), 91-96 (1988).</li><li>Hamir, A. N., Moser, G., Rupprecht, C. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+five+year+(1985-1989)+retrospective+study+of+equine+neurological+diseases+with+special+reference+to+rabies.">A five year (1985-1989) retrospective study of equine neurological diseases with special reference to rabies.</a> Journal of Comparative Pathology. 106 (4), 411-421 (1992).</li><li>Inoue, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cross-reactive+antigenicity+of+nucleoproteins+of+lyssaviruses+recognized+by+a+monospecific+antirabies+virus+nucleoprotein+antiserum+on+paraffin+sections+of+formalin-fixed+tissues.">Cross-reactive antigenicity of nucleoproteins of lyssaviruses recognized by a monospecific antirabies virus nucleoprotein antiserum on paraffin sections of formalin-fixed tissues.</a> Pathology International. 53 (8), 525-533 (2003).</li><li>Webster, J. D., Miller, M. A., Dusold, D., Ramos-Vara, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+prolonged+formalin+fixation+on+diagnostic+immunohistochemistry+in+domestic+animals.">Effects of prolonged formalin fixation on diagnostic immunohistochemistry in domestic animals.</a> Journal of Histochemistry and Cytochemistry. 57 (8), 753-761 (2009).</li><li>Feiden, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunohistochemical+staining+of+rabies+virus+antigen+with+monoclonal+and+polyclonal+antibodies+in+paraffin+tissue+sections.">Immunohistochemical staining of rabies virus antigen with monoclonal and polyclonal antibodies in paraffin tissue sections.</a> Zentralblatt fur Veterinarmedizin Reihe B. 35 (4), 247-255 (1988).</li><li>Manning, S. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+rabies+prevention--United+States,+2008:+recommendations+of+the+Advisory+Committee+on+Immunization+Practices.">Human rabies prevention--United States, 2008: recommendations of the Advisory Committee on Immunization Practices.</a> Morbidity and Mortality Weekly Reports Recommendations and Reports. 57, 1-28 (2008).</li><li>WHO. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Laboratory techniques in rabies. 1, 67-72 (2018).</li><li>Patrick, E. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhanced+Rabies+Surveillance+Using+a+Direct+Rapid+Immunohistochemical+Test.">Enhanced Rabies Surveillance Using a Direct Rapid Immunohistochemical Test.</a> Journal of Visualized Experiments. (146), (2019).</li><li>Whitfield, S. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+comparative+study+of+the+fluorescent+antibody+test+for+rabies+diagnosis+in+fresh+and+formalin-fixed+brain+tissue+specimens.">A comparative study of the fluorescent antibody test for rabies diagnosis in fresh and formalin-fixed brain tissue specimens.</a> Journal of Virology Methods. 95 (1-2), 145-151 (2001).</li><li>WHO. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Diagnostic procedures for antigen detection. , (2016).</li><li>Jarvis, J. A., Franke, M. A., Davis, A. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rabies+direct+fluorescent+antibody+test+does+not+inactivate+rabies+or+eastern+equine+encephalitis+viruses.">Rabies direct fluorescent antibody test does not inactivate rabies or eastern equine encephalitis viruses.</a> Journal of Virology Methods. 234, 52-53 (2016).</li></ol>

The authors have nothing to disclose.

Due to the high fatality rate of rabies after the symptom onset, the diagnosis of suspect animals for RABV infection is extremely critical for an appropriate post-exposure prophylactic treatment. Rabies diagnosis primarily depends on DFA, DRIT, and PCR-based techniques using fresh or frozen tissues. For testing of formalin-fixed tissues, the IHC test provides an alternative method for the sensitive and specific detection of RABV antigen. While the tissues fixed in formalin have proteins stabilized due to the modification...

Rabies is an acute progressive encephalitis caused by the negative sense RNA viruses belonging to the genus lyssavirus1. Nearly 99% of all human deaths caused by the infection with rabies virus (RABV), the type member of the genus, is transmitted by dogs2. Rabies diagnosis of suspect animals relies on the detection of antigen (primarily viral encoded nucleoprotein, N protein) in complex with genomic RNA (ribonucleoprotein complex, RNP) in the brain tissue3. The antigen detection by the direct fluorescent antibody (DFA) test is considered the gold standard for rabies diagnosis4. The method utilizes fresh or fresh frozen brain material, a touch impression on a slide, fixation in acetone, staining using commercially available fluorescent isothiocyanate (FITC) labeled monoclonal or polyclonal antibodies (mAbs/pAbs) and read by the fluorescence microscopy5. The DFA test is rapid, sensitive, and specific for rabies antigen detection in fresh brain tissue. Recently, a direct rapid immunohistochemical test (DRIT), modified immunohistochemistry (IHC) technique, was demonstrated to exhibit similar sensitivity to DFA but offers the advantage of light microscopy for visualization6. While the detection method used in DRIT, is similar to IHC, the initial step utilizes fresh or frozen tissues to generate touch impressions of the sample followed by fixation in formalin.
IHC is a widely used technique to determine histological changes and detection of proteins using specific antibodies in formalin-fixed tissues embedded in paraffin blocks. IHC is an established alternative test for the rabies antigen detection in the tissue sections7. IHC has been particularly utilized for the diagnosis of retrospective cases that exhibited neurological diseases to determine the burden of rabies8. Paraffin-embedded formalin-fixed tissues preserve the proteins for the detection even after several years when stored at ambient temperature9. Formalin treatment modifies proteins by cross-linking and altering the amino acid side chains, which might make the epitopes no longer reactive against antibodies10. While the IHC test for rabies antigen detection involves either mAbs or pAbs, the latter is advantageous as multiple epitopes and divergent lyssaviruses can be detected11.
The standard steps involved in IHC are formalin fixation of tissues, embedding in paraffin blocks, sectioning of tissues, deparaffinization and hydration, epitope recovery, reactivity against primary and secondary antibodies, and the development using chromogenic substrates. This manuscript describes a detailed account of the protocol for rabies diagnosis. For rabies antigen detection, mouse serum immunized with RABV (pAbs) generated at the U.S. Centers for Disease Control and Prevention (CDC) Atlanta, Georgia, in combination with biotinylated anti-mouse secondary antibodies are utilized. Biotinylated Abs are detected by the addition of streptavidin-horseradish peroxidase (HRP) complex followed by the color development with amino-ethylcarbazole substrate.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>3% hydrogen peroxide</td>
			<td>Pharamacy brands</td>
			<td></td>
			<td>Off the shelf 3% H2O2</td>
		</tr>
		<tr>
			<td>3-Amino-9-ethylcarbazole (AEC)</td>
			<td>Millipore Sigma</td>
			<td>A6926</td>
			<td></td>
		</tr>
		<tr>
			<td>Acetate Buffer pH 5.2</td>
			<td>Poly Scientific R&#38;D Corp.</td>
			<td>s140</td>
			<td></td>
		</tr>
		<tr>
			<td>Buffered Formalin 10% Phosphate Buffered</td>
			<td>Fisher Scientific</td>
			<td>SF100-4</td>
			<td>Certified</td>
		</tr>
		<tr>
			<td>Cover slips Corning</td>
			<td>Fisher Scientific</td>
			<td>12-553-471</td>
			<td>24 X 50 mm</td>
		</tr>
		<tr>
			<td>Ethanol 190 Proof</td>
			<td>Pharmco-AAPER</td>
			<td>111000190</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol 200 Proof</td>
			<td>Pharmco-AAPER</td>
			<td>111000200</td>
			<td></td>
		</tr>
		<tr>
			<td>Gill&#39;s hematoxylin formulation #2</td>
			<td>Fisher Scientific</td>
			<td>CS401-1D</td>
			<td></td>
		</tr>
		<tr>
			<td>HistoMark Biotin-Streptavidin Peroxidase Kit</td>
			<td>seracare</td>
			<td>71-00-18</td>
			<td>Mouse Primary Antibody&#160;</td>
		</tr>
		<tr>
			<td>ImmunoHistoMount</td>
			<td>Millipore Sigma</td>
			<td>i1161</td>
			<td>Mounting media</td>
		</tr>
		<tr>
			<td>N,N, Dimethyl formamide GR</td>
			<td>Fisher Scientific</td>
			<td>D119</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate Buffered Saline&#160;</td>
			<td>HyClone</td>
			<td>RR14440.01</td>
			<td>01M, pH 7.2 (pH 7.2-7.6)</td>
		</tr>
		<tr>
			<td>Plan-APOCHROMAT 40X/0.95 Objective</td>
			<td>Multiple vendors</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Plan-APOCHROMATIC 20X/0.75 Objective</td>
			<td>Multiple vendors</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Pronase</td>
			<td>Millipore Sigma</td>
			<td>53702</td>
			<td>Protease, Streptomyces griseus</td>
		</tr>
		<tr>
			<td>Scott&#39;s Tap Water&#160;</td>
			<td>Poly Scientific R&#38;D Corp.</td>
			<td>s1887</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue-Tek Slide stain set</td>
			<td>Fisher Scientific</td>
			<td>50-294-72</td>
			<td></td>
		</tr>
		<tr>
			<td>TWEEN-80&#160;</td>
			<td>Millipore Sigma</td>
			<td>P1754</td>
			<td></td>
		</tr>
		<tr>
			<td>Xylene</td>
			<td>Fisher Scientific</td>
			<td>X3S-4</td>
			<td>Histological Grade</td>
		</tr>
		<tr>
			<td>Zeiss Axioplan 2 imaging - microscope</td>
			<td>Multiple vendors</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

immunohistochemistry test for the lyssavirus antigen detection from formalin-fixed tissues

One of the primary diagnostic modalities for rabies is the detection of viral ribonucleoprotein (RNP) complex (antigen) in the infected tissue samples. While the direct fluorescent antibody (DFA) test or the direct rapid immunohistochemical test (DRIT) are most commonly utilized for the antigen detection, both tests require fresh and/or frozen tissues for impressions on slides prior to the antigen detection using antibodies. If samples are collected and fixed in formalin, neither test is optimal for the antigen detection, however, testing can be performed by conventional immunohistochemistry (IHC) after embedding in paraffin blocks and sectioning. With this IHC method, tissues are stained with anti-rabies antibodies, sections are deparaffinized, antigen retrieved by partial proteolysis or other methods, and incubated with primary and secondary antibodies. Antigens are stained using horseradish peroxidase / amino ethyl carbazole and counterstained with hematoxylin for the visualization using a light microscope. In addition to the specific antigen detection, formalin fixation offers other advantages like the determination of histological changes, relaxed conditions for specimen storage and transport (under ambient temperatures), ability to test retrospective cases and improved biological safety through the inactivation of infectious agents.

Figure 2 demonstrates representative IHC staining results of positive and negative control samples in different brain tissues tested. Figure 2A,D,G represent positive samples at 200x, while Figure 2B,E,H correspond to 400x magnification, respectively. Figure 2A-C correspond to the brainstem; Figure 2...

Watch this Scientific Journal Video about Immunohistochemistry Test for the Lyssavirus Antigen Detection from Formalin-Fixed Tissues at JoVE.com

Immunohistochemistry Test for the Lyssavirus Antigen Detection from Formalin-Fixed Tissues

Here, we present an immunohistochemistry test protocol for the detection of rabies virus antigen as an alternative diagnostic test for formalin-fixed tissues.

One of the primary diagnostic modalities for rabies is the detection of viral ribonucleoprotein (RNP) complex (antigen) in the infected tissue samples. ...

Tissues used for Immunohistochemistry and for diagnostic histopathology are routinely fixed in 10%phosphate buffer formalin. The detection of rabies virus antigen in brain tissue that has been fixed in formalin presents a unique challenge, and the IHC test has been found to be a sensitive and specific method for rabies virus antigen detection. The primary advantage of biopsy or other detection methods is that antigens can be detected in relation to tissue and cellular morphology.With IHC, localization of rabies antigen in brain and non-brain tissues, provide additional information that could not be obtained through routine Hematoxylin and Eosin stain sections. Rabies diagnostic testing is important for the initiation of post-exposure prophylaxis in humans exposed to rabies virus. IHC is one of the most commonly performed assays on formalin-fixed tissues.With specific antibodies, it can be used for the detection of respective pathogens. To begin this procedure, place three to five millimeter pieces of brain tissue, collected after necropsy, into a 10%buffered formalin solution for 24 to 72 hours. Record the approximate tissue weight, tissue type, and the volume of formalin.Place the tissues in 70%ethanol for longer tissue storage after formalin fixation, and prior to processing. Following the specimen fixation, dissect the tissue to include the important brain areas, such as cross sections of the brainstem, the cerebellum, or the hippocampus. Each, cut to a thickness between three and five millimeters.Place the tissues into processing cassettes. Process the tissue cassettes for paraffin wax infiltration. Embed them into paraffin blocks and section them on a microtome.First set up the staining dish as outlined in Figure 1 of the text protocol. Fill each dish with 250 milliliters of the prepared solution. To prepare the AEC substrate stock solution, use a glass pipette to dissolve one 20 milligram tablet of AEC in 5 milliliters of N, N-Dimethylformamide.To prepare the protease stock solution, dissolve 7 milligrams of the protease in 200 milliliters of PBS. To prepare the rinse buffer, add 100 milliliters of Tween 80 to 990 milliliters of PBS, and mix well to form a homogenous solution of PBT-T. Using a microtome, make a five micrometer paraffin section.Load the section on a water bath at 38 degrees Celsius and collect it onto glass slides. Label the slides with a reagent resistant pen. Place the slides onto a tray and melt in an oven at 55 to 60 degrees Celsius for one hour.Then, remove the slides from the oven and immediately deparaffinize them, using 3 consecutive xylene rinses of 5 minutes each. After this, rehydrate the sections on the slide by sequential immersions in decreasing dilutions of ethanol to deionized water, as outlined in the text protocol. Treat the slides with the protease for 30 minutes for proteolytic antigen retrieval, then rinse the slides in PBS-T for 10 minutes, and treat them with 3%hydrogen peroxide for 10 minutes.After this, wash the slides again with PBS-T for 10 minutes. To begin, remove one slide from the buffer, making sure to only handle one slide at a time, while the others remain submerged, and use a paper towel to blot off excess buffer from around the tissue section. Place the slides into a humidity chamber and incubate with normal goat serum at room temperature for 15 minutes.Next, incubate the slides in the humidity chamber with the optimal predetermined dilution primary anti-rabies antibody, and negative control antibodies, at room temperature for 60 minutes with no washes in between. After this, wash the slides with PBS-T for 10 minutes and incubate in a humidity chamber with the biotinylated antibody at room temperature for 15 minutes. Wash the slides again with PBS-T for 10 minutes.Incubate the slides and the humidity chamber with strip divide and HRB complex at room temperature for 15 minutes and wash the PBS-T for 10 minutes. Then, incubate with AEC in a humidity chamber at room temperature for 10 minutes. Wash the slides in deionized water for 10 minutes and counterstain with Gill's Hematoxilyn, diluted one to two with deionized water for two minutes.After this, rinse off the excess hematoxylin by dip rinsing the slides in deionized water. Rinse the slides in Scott's Tap Water for 30 seconds and wash in deionized water for 10 minutes. Remove the slides one at a time and mount them with water-soluble mounting medium.Then, use a light microscope to read the slides. In this study, an immunocytic chemistry test is demonstrated for the detection of rabies virus antigen as an alternative diagnostic test for formalin-fixed tissues. A representative immunocytic chemistry staining result of positive and negative control samples in different brain tissues is shown here, including the brainstem, the cerebellum and purkinje cells, and the hippocampus.The magenta red staining, which indicates the color development by using AEC substrate against the blue counterstained background, is due to the reactivity of antibodies against the rabies antigen. A positive result in Immunohistochemistry corresponds to the magenta red staining in the tissue sections. The staining of cytoplasmic inclusions and granular inclusions of varying size are indicative of samples positive for RABV infections.Samples are deemed negative if no specific red staining or only the blue background, due to hematoxylin, were observed. In addition to the positive staining, the distribution of inclusions could provide indirect quantification of levels of rabies antigen in the sample, which might correspond to the viral load of the tissue sample. Irrespective of the levels of distribution, any specific staining will classify the sample as positive for RABV antigen detection.Tissues should be fixed adequately in formalin, and avoid freezing the tissues during this process. Rabies antigen is detected by IHC, then molecular techniques can be performed on paraffin sections to determine the Lyssavirus variant based on RNA sequence information. Reagents like xylene and formalin should be handled in fume hoods.Care should be taken while handling AEC as it is a carcinogen.

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4.1K Views.  Centers for Disease Control and Prevention. Tissues used for Immunohistochemistry and for diagnostic histopathology are routinely fixed in 10%phosphate buffer formalin. The detection of rabies virus antigen in brain tissue that has been fixed in formalin presents a unique challenge, and the IHC test has been found to be a sensitive and specific method for rabies virus antigen detection. The primary advantage of biopsy or other detection methods is that antigens can be detected in relation to tissue and cellular morphology.With I...